CN106190948B - CHO-S cell semisolid culture medium and preparation method and application thereof - Google Patents
CHO-S cell semisolid culture medium and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a CHO-S cell semisolid culture medium and a preparation method and application thereof. The semi-solid culture medium comprises the following components in percentage by weight: 2.46 to 2.48 percent of CD FortiCHO culture medium and 2 to 3 percent of methylcellulose. The CHO-S cell semisolid culture medium is prepared by fully and uniformly mixing 2 XCD FortiCHO and 2 Xmethylcellulose solution according to the volume ratio of 1:1, centrifuging to remove particles invisible to naked eyes, and collecting supernatant centrifugate. The CHO-S cell semisolid culture medium can be used for replacing an imported CHO-S cell semisolid culture medium to culture CHO-S cells, the cells grow, the protein expression amount is high, the growth state is good, and the research and development production cost can be greatly reduced.
Description
Technical Field
The invention relates to a semisolid culture medium suitable for CHO-S cell growth, a preparation method and application thereof, and belongs to the development field of CHO-S cell strains.
Background
The target specificity and high expression level of the antibody have important significance in clinic, so that how to find a cell line which can stably secrete a large amount of correct antibody in one step more quickly than a competitor is very important. For this reason, the traditional cell screening method adopts a limiting dilution method or a cell sorting method, the workload is large, the time consumption is long, generally, the screening of 1000 cells takes 8 weeks, and a good clone can be obtained finally; and mechanical damage is easily caused to cells when choosing clone, so that the survival rate of clone is reduced. At present, enterprises often use a semi-solid culture medium method for monoclonal screening. The cell grows in a semi-solid culture medium to form a monoclonal cell mass, the antibody is secreted around the cell mass, and the semi-solid culture medium contains a fluorescence-labeled secondary antibody which can be specifically combined with the Fc fragment of the antibody and can emit fluorescence when being excited by light with a certain wavelength. The size and fluorescence intensity of the monoclonal cell mass can be measured by using image analysis software, so that the monoclonal cell mass with vigorous growth and high expression level can be screened. The Clone Pix system screening method developed by Molecular Device company (refer to the research on CHO cell strain development technical strategy) effectively avoids the defect of manually screening cells, can select a large number of clones in a short time, is based on a fluorescence detection technology but does not need an expression vector to generate fluorescence, thereby effectively avoiding false positive; the cells are in the semi-solid culture medium, the selection process is soft, the survival rate of the cells is increased, and therefore the efficiency is greatly improved.
In CHO cell Clone screening, a semi-solid medium suitable for CHO cell growth is one of the most critical elements, as exemplified by Clone Pix system screening method. Molicular Devices developed a semi-solid medium suitable for the growth of various cells, in which Clonemedia-CHO-G (Cat: K8740), a semi-solid medium suitable for CHO-S cells, was very expensive, sold at a price of 90mL of 4251 yuan, had a long shelf life, high logistics costs, and few alternatives to this product, and thus the development costs for development of CHO-S cells were very high. Besides the fixed components, the preparation method of the semi-solid medium plays an important role in cell growth. The formulation and preparation method of CloneMedia-CHO-G semisolid culture medium are trade secrets of molecular Devices and are not known to the public.
Therefore, there is a need to develop a new semi-solid culture medium suitable for the growth of CHO-S cells and a preparation method thereof, so as to reduce the scientific research cost and increase the freedom of choice of experimental materials.
Disclosure of Invention
In order to solve the above problems, it is an object of the present invention to provide a CHO-S cell semisolid culture medium which is suitable for the growth of CHO-S cells, can be used in the cell clone screening stage, and can effectively replace Clonemedia-CHO-G semisolid culture medium suitable for CHO-S cells developed by Molicula plants.
Another object of the present invention is to provide a method for preparing a semisolid culture medium suitable for CHO-S cells.
In order to solve the problems, the technical scheme of the invention is as follows:
in one aspect, the invention provides a CHO-S cell semisolid culture medium, which comprises the following components in percentage by weight: 2.46 to 2.48 percent of CD FortiCHO culture medium and 2 to 3 percent of methylcellulose.
According to a specific embodiment of the invention, the CHO-S cell semisolid culture medium is obtained by fully and uniformly mixing 2 XCDFortCHO and 2 Xmethylcellulose solution according to the volume ratio of 1:1, centrifuging to remove particles invisible to naked eyes, and collecting supernatant centrifugate.
Preferably, the above-mentioned CD FortiCHO medium is a solid medium CD FortiCHO AGT mediumPrototype or CD FortiCHO II, which are commercially available from the company gibco.
Preferably, the methyl cellulose has a viscosity of 25cps to 1500cps at 20 ℃ at 2% by mass to volume ratio.
In one embodiment of the present invention, the methylcellulose has an average molecular weight of 17000-63000.
Preferably, the content of the methyl cellulose is 2.7-3.0%.
In another aspect, the present invention also provides a method for preparing the semi-solid culture medium for CHO-S cells, which comprises:
a. fully and uniformly mixing 2 XCD FortiCHO culture medium and 2 Xmethylcellulose solution in a volume ratio of 1: 1;
b. centrifuging to remove invisible particulate matter, and collecting supernatant centrifugate to obtain CHO-S cell semisolid culture medium; wherein, the centrifugation condition is preferably 4000 rpm-10000 rpm, and the centrifugation is more than 30 min.
According to a specific embodiment of the invention, the 2 × CD FortiCHO medium is prepared according to the following method: weighing the CD FortiCHO culture medium, adding ultrapure water, fully stirring and dissolving, and filtering and sterilizing by a 0.20 mu M filter membrane to obtain the 2 XCD FortiCHO culture medium.
According to a specific embodiment of the present invention, in the present invention, the 2 x methyl cellulose solution is prepared according to the following method: weighing methylcellulose, adding ultrapure water, fully stirring and dissolving, and autoclaving to obtain 2 Xmethylcellulose solution. The autoclaving conditions can be conventional medium sterilization conditions, for example autoclaving at about 121 ℃ for 30 min.
The preparation method of the semi-solid culture medium is generally characterized in that the culture medium is clear and transparent through visual observation, and is judged to be uniformly stirred and all components are fully dissolved without solid particles. The components of the semi-solid culture medium are generally ensured to be fully dissolved by prolonging the stirring time or increasing the stirring strength or boiling and the like. However, the semi-solid culture medium prepared by the above-mentioned conventional method in the art is not optimal in practical use, and the application effect is lower than that of the commercial semi-solid culture medium. Under the teaching of textbooks, a person skilled in the art generally thinks that the semisolid culture medium is fully stirred and dissolved, and is clarified by visual observation, and further does not explore the influence of the microscopic morphology on the application effect, and related scientific and technical documents or patent documents are not searched.
The inventor accidentally places the semi-solid culture medium under an optical microscope for observation, and observes that a large amount of fine insoluble solid particles still exist in the semi-solid culture medium without the solid particles through naked eye observation. Further studies found that centrifugation removed these solid particles and the centrifuged CHO-S cell semisolid medium was used for CHO-S cell culture. The test result shows that: compared with a semi-solid culture medium which is not subjected to centrifugal treatment, the semi-solid culture medium subjected to centrifugal treatment has the advantages that CHO-S cells grow, the protein expression amount is high, the growth state is relatively good, and the subsequent cell screening work is benefited. The centrifugation of the CHO-S cell semisolid culture medium for culturing CHO-S cells has unexpected technical effects beyond the expectation of those skilled in the art.
The criterion of the centrifugation condition is that the semisolid culture medium is observed to be free of fine solid particles under an optical microscope (i.e., no visible solid particles are observed under an optical microscope). Preferably, the invention recommends that the centrifugation condition is 4000 rpm-10000 rpm, and the centrifugation is more than 30 min. The more specific speed and time of centrifugation can be selected by the skilled person depending on the specific experimental conditions, centrifuge rotor size. Preferably, the centrifugation is preferably performed at low temperature, such as 4 ℃. In particular to the centrifugal operation containing the CD FortiCHO culture medium which is operated at the lowest temperature of 4 ℃.
In another aspect, the invention also provides the application of the CHO-S cell semisolid culture medium in the culture of CHO-S cells. In specific application, the culture conditions for culturing the CHO-S cells are as follows: 5% CO236.5 ℃. Preferably, the culture is a static incubator culture or a 120rpm shake flask culture. The test result shows that: the CHO-S cell semisolid culture medium is used for culturing CHO-S cells, the CHO-S cells grow, the protein expression amount is high, the growth state is relatively good, and the CHO-S cell semisolid culture medium is beneficial to subsequent cell screening work.
The invention has the beneficial effects that:
the CHO-S cell semisolid culture medium provided by the invention is suitable for the growth of CHO-S cells, can be used in a cell cloning and screening stage, and provides more choices for researchers or enterprises; the culture medium CloneMedia-CHO-G can be used for replacing a CHO-S cell semi-solid culture medium of the molecular Devices company, so that the influence of overlong supply period of imported products on scientific research work is reduced, and the dependence of enterprises on the products is reduced; 90mL of Clonemedia-CHO-G is sold at 4251 yuan, and the cost for preparing the semi-solid culture medium with the same volume is about 130 yuan, so that the development and production cost can be greatly reduced by adopting the semi-solid culture medium to replace a CHO-S cell semi-solid culture medium Clonemedia-CHO-G of molecular Devices company; the CHO-S cell semisolid culture medium prepared by the method for preparing the CHO-S cell semisolid culture medium is used for cell culture, the cells grow, the protein expression amount is high, the growth state is good, and an unexpected effect is obtained and is beyond the expectation of ordinary technicians in the field; the preparation method provided by the invention is simple to operate and strong in feasibility, and can be used for preparing the semi-solid culture medium in batches by combining a high-capacity centrifuge so as to meet the research and development production requirements.
Drawings
FIG. 1 is a 50-fold magnification of an unformulated CHO-S cell semisolid culture medium formulated in example 1.
FIG. 2 is a 50-fold magnification of a self-prepared CHO-S cell semisolid culture medium, and the formula of the culture medium is shown in example 1.
FIG. 3 is a 50-fold magnification of the non-centrifuged semi-solid media for self-prepared CHO-S cells, and the media formulation is given in example 2.
FIG. 4 is a 50-fold magnification of centrifugation of self-prepared CHO-S cell semisolid culture medium, the medium formulation is shown in example 2.
FIG. 5 is a white light plot, at 50X magnification, of the sixth day of growth state of clones from reconstituted CHO-S cell semi-solid medium (not centrifuged) according to example 1.
FIG. 6 is a fluorescence plot at 50X magnification of a sixth day of growth state of clones prepared from CHO-S cell semisolid medium (not centrifuged) according to the medium formulation of example 1.
FIG. 7 is a white light map, 50-fold magnification, of the sixth day of growth state of self-formulated CHO-S cell semisolid medium (centrifuged) clones, see example 1 for medium formulation.
FIG. 8 is a fluorescence plot at 50X magnification of a sixth day of growth state of self-conditioned CHO-S cell semisolid medium (centrifuged) clones, see example 1 for medium formulation.
FIG. 9 is a white light map, 50-fold magnification, of the sixth day of growth state of self-formulated CHO-S cell semisolid medium (centrifuged) clones, see example 2 for medium formulation.
FIG. 10 is a fluorescence plot at 50X magnification of a sixth day of growth state of self-conditioned CHO-S cell semisolid medium (centrifuged) clones, the medium formulation of which is given in example 2.
FIG. 11 is a white light map, 50-fold magnification, of the sixth day of growth state of self-formulated CHO-S cell semisolid medium (centrifuged) clones, see example 4 for medium formulation.
FIG. 12 is a fluorescence plot at 50X magnification of a sixth day of growth state of self-conditioned CHO-S cell semisolid medium (centrifuged) clones, see example 4 for medium formulation.
FIG. 13 is a white light plot at 50X magnification of the sixth day of growth state of self-conditioned CHO-S cell semisolid medium (centrifuged) clones, see example 6 for medium formulation.
FIG. 14 is a fluorescence plot at 50X magnification of a sixth day of growth state of self-conditioned CHO-S cell semisolid medium (centrifuged) clones, the medium formulation of which is given in example 6.
FIG. 15 is a white light plot at 50 times magnification of the sixth day growth state of commercial CHO-S cell semi-solid medium Clonemedia-CHO-G clone.
FIG. 16 is a fluorescence plot at 50 times magnification of the sixth day growth state of commercial CHO-S cell semi-solid medium Clonemedia-CHO-G clone.
Detailed Description
The technical solutions of the present invention, the characteristics thereof, and the technical effects thereof in the applications are further described in detail by the following specific examples, but the present invention is not limited thereby.
Example 1 CHO-S cell semisolid Medium formulation
Materials:
CD FortiCHO AGT Medium Prototype, supplier Gibco, cat # A14286 EF;
methylcellulose available from Sigma under the trade designation M0262-500G, average molecular weight 41000, 2% viscosity of the methylcellulose is 400cPs at 20 ℃.
According to the weight percentage: 2.46% CD FortiCHO AGT Medium Prototype, 2% methylcellulose.
Example 2 CHO-S cell semisolid Medium formulation
CD FortiCHO AGT Medium Prototype, supplier Gibco, cat # A14286 EF;
methylcellulose available from Sigma under the trade designation M0262-500G, average molecular weight 41000, 2% viscosity of the methylcellulose is 400cPs at 20 ℃.
According to the weight percentage: 2.46% CD FortiCHO AGT Medium Prototype, 3% methylcellulose.
Example 3 CHO-S cell semisolid Medium formulation
CD FortiCHO AGT Medium Prototype, supplier Gibco, cat # A14286 EF;
methylcellulose available from Sigma under the trade designation M0262-500G, average molecular weight 41000, 2% viscosity of the methylcellulose is 400cPs at 20 ℃.
According to the weight percentage: 2.46% of CD Fortico AGT Medium Prototype, 2.7% of methylcellulose.
Example 4 CHO-S cell semisolid Medium formulation
CD FortiCHO II powder, supplier Gibco, cat # A24363 SA;
methylcellulose available from Sigma under the trade designation M0262-500G, average molecular weight 41000, 2% viscosity of the methylcellulose is 400cPs at 20 ℃.
According to the weight percentage: 2.48% CD FortiCHO II, 2% methylcellulose.
Example 5 CHO-S cell semisolid Medium formulation
CD FortiCHO II powder, supplier Gibco, cat # A24363 SA;
methylcellulose available from Sigma under the trade designation M0262-500G, average molecular weight 41000, 2% viscosity of the methylcellulose is 400cPs at 20 ℃.
According to the weight percentage: 2.48% CD FortiCHO II, 2.7% methylcellulose.
Example 6 CHO-S cell semisolid Medium formulation
CD FortiCHO II powder, supplier Gibco, cat # A24363 SA;
methylcellulose available from Sigma under the trade designation M0262-500G, average molecular weight 41000, 2% viscosity of the methylcellulose is 400cPs at 20 ℃.
According to the weight percentage: 2.48% CD FortiCHO II, 3% methylcellulose.
Example 7 CHO-S cell semisolid Medium formulation
CD FortiCHO AGT Medium Prototype, supplier Gibco, cat # A14286 EF;
methylcellulose available from Sigma under the trade designation M6385-500G, average molecular weight 17000, 2% viscosity of the methylcellulose at 20 ℃ is 25 cPs.
According to the weight percentage: 2.46% CD FortiCHO AGT Medium Prototype, 3% methylcellulose.
Example 8 CHO-S cell semisolid Medium formulation
CD FortiCHO AGT Medium Prototype, supplier Gibco, cat # A14286 EF;
methylcellulose available from Sigma under the trade designation M0387-500G, average molecular weight 63000, and a viscosity of 1500cPs at 2% at 20 ℃.
According to the weight percentage: 2.46% CD FortiCHO AGT Medium Prototype, 2% methylcellulose.
Example 9 CHO-S cell semisolid Medium formulation
CD FortiCHO II powder, supplier Gibco, cat # A24363 SA;
methylcellulose available from Sigma under the trade designation M6385-500G, average molecular weight 17000, 2% viscosity of the methylcellulose at 20 ℃ is 25 cPs.
According to the weight percentage: 2.48% CD FortiCHO AGT Medium Prototype, 3% methylcellulose.
Example 10 CHO-S cell semisolid Medium formulation
CD FortiCHO II powder, supplier Gibco, cat # A24363 SA;
methylcellulose available from Sigma under the trade designation M0387-500G, average molecular weight 63000, and a viscosity of 1500cPs at 2% at 20 ℃.
According to the weight percentage: 2.48% CD FortiCHO II, 2% methylcellulose.
Example 11 preparation of CHO-S cell semisolid Medium
The CHO-S cell semisolid culture medium is prepared according to the following steps:
(1)2 × CD FortiCHO medium preparation: CD FortCHO AGT Medium Prototype or CD FortCHO II solid Medium is accurately weighed, ultrapure water is added, a magnetic stirrer at 400rpm is used for stirring for 30min to fully dissolve the Medium, then a 0.20 mu M filter membrane (sartolab-P20plus, cat: 18056) is used for filtration sterilization to obtain 2 XCDFortCHO Medium, and the Medium is stored in a refrigerator at 4 ℃ for standby.
(2)2 × methylcellulose solution preparation: accurately weighing methylcellulose, adding purified water, stirring with a magnetic stirring vessel at 400rpm for 60min to dissolve, autoclaving at 121.3 deg.C for 20 min, cooling to room temperature, and storing in a refrigerator at 4 deg.C for more than 30 min.
(3) The prepared 2 XCD FortiCHO culture medium and 2 Xmethylcellulose solution are mixed in a volume ratio of 1:1 in a biosafety cabinet and stirred overnight with a magnetic stirrer at 600rpm at 4 ℃.
(4) And (3) respectively packaging the semi-solid culture medium obtained in the step (3) in a 50mL sterile centrifuge tube, centrifuging at 4000rpm for 30min until the supernatant is observed to have no fine insoluble solid particles under a microscope at a magnification of 50 times, discarding the solid culture medium (about 5mL) at the bottom layer of the centrifuge tube, and collecting the upper semi-solid culture medium for later use in a refrigerator at the temperature of-20 ℃.
Example 12 CHO-S cell semi-solid Medium (centrifuged and not centrifuged) growth State alignment of CHO-S cells cultured in Clonemedia-CHO-G, a commercial CHO-S cell semi-solid Medium
Test materials:
(1) example 1 CHO-S cell semi-solid Medium, formulated according to steps 1-4 described in example 11, to give a centrifuged self-formulated CHO-S cell semi-solid medium.
(2) Example 1 CHO-S cell semi-solid Medium, formulated according to steps 1-3 described in example 11, to give an uncreped, self-formulated CHO-S cell semi-solid medium.
(3) Example 2 CHO-S cell semi-solid Medium, formulated according to steps 1-4 described in example 11, to give a centrifuged self-formulated CHO-S cell semi-solid medium.
(4) Example 4 CHO-S cell semi-solid Medium, formulated according to steps 1-4 described in example 11, to give a centrifuged self-formulated CHO-S cell semi-solid medium.
(5) Example 6 CHO-S cell semi-solid Medium, formulated according to steps 1-4 described in example 11, to give a centrifuged self-formulated CHO-S cell semi-solid medium.
(6) Commercial CHO-S cell semisolid Medium Clonemedia-CHO-G, molecular Devices, Cat: K8740.
(7) CHO-S host cells, Shanghai Jinmant Biotech, Inc., express IgG.
The test method comprises the following steps:
the CHO-S cell semisolid culture medium (both centrifuged and not centrifuged) and the molecular Devices commercial CHO-S cell semisolid culture medium provided by the present invention were plated with CHO-S host cells and compared for cell growth and IgG protein expression.
The prepared semi-solid culture medium and commercial culture medium were taken out from a-20 deg.C refrigerator, and placed in a 4 deg.C refrigerator overnight to dissolve completely. If one is spread on a 6-well plate, 15ml of semi-solid medium is added to a 50ml sterile centrifuge tube in a cell house clean bench, followed by 0.6ml of L-Glutamine (Gibco, 200mM, cat: 25030 081), followed by 150. mu.l of CloneDetect (molecular Devices, cat: K8200), which are then mixed well, followed by 3000-12000 cells (i.e., 500-2000 cells per well), mixed well again, and 2ml of the mixture is added to each well (avoiding steam generation and tiling). Finally placing the mixture into a constant temperature incubator (36.5 ℃, 5% CO)2) The culture was carried out for 6 days.
And (3) test results: see fig. 1-16.
FIGS. 1 and 2, and FIGS. 3 and 4 are 50-fold enlarged views of the centrifuged and non-centrifuged liquid of the self-prepared CHO-S cell semisolid culture medium. The results show that a large amount of fine insoluble solid particles still exist in the non-centrifugal CHO-S cell semisolid culture, and the actual particle size of the particles is about 10-50 mu m.
In terms of the growth state of CHO-S cells cultured in the non-centrifuged and centrifuged CHO-S cell self-prepared semisolid culture medium (see fig. 5-14), the non-centrifuged CHO-S cell self-prepared semisolid culture medium has more background impurities (see fig. 5 and 6), the cell growth is relatively small, the IgG protein expression is low, and the growth state is relatively poor; the centrifuged CHO-S cell self-prepared semisolid culture medium has a clear background, large cell growth, high IgG protein expression level (the diameter of an antibody fluorescence pattern expressed by cell clones is about 1-2mM), and relatively good growth state (FIG. 7-FIG. 14).
Centrifugal self-prepared CHO-S cell semisolid culture medium (figure 7-figure 14)) compared with the CHO-S cell growth state (figure 15, figure 16) cultured by a commercial CHO-S cell semisolid culture medium, the centrifugal self-prepared semisolid culture medium CHO-S cell clone grows more compactly, and the fluorescence intensity and the size part clone performance are stronger.
The test results show that the application effect of the centrifugal self-prepared CHO-S cell semisolid culture medium provided by the invention is basically up to or even better than that of a commercial semisolid culture medium of molecular Devices; the preparation method of the semi-solid culture medium provided by the invention is beneficial to improving the application performance of the semi-solid culture medium.
Example 13 centrifugation of Ready-to-prepare CHO-S cell semi-solid Medium with commercial CHO-S cell semi-solid Medium Clonemedia-CHO-G Fed-batch CHO-S cells
The test method comprises the following steps: the CHO-S cell semisolid culture medium centrifugation liquid prepared in example 12 (see example 1 for the culture medium formulation) and the commercial culture medium (same as example 12) were separately removed from the refrigerator at-20 deg.C and dissolved overnight in the refrigerator at 4 deg.C. A6-well plate was laid on the cell house superclean bench: a50 ml sterile centrifuge tube was filled with 15ml of semi-solid medium, followed by 0.6mlL-Glutamine (Gibco, 200mM, cat: 25030-. Finally placing the mixture into a constant temperature incubator (36.5 ℃, 5%CO2) The culture was carried out for 6 days. Selecting clones to be cultured in 96-well for 5 days, selecting certain clones from 96-well to be cultured in 24-well for 5 days in the Elisa titer high-low and growth state, selecting certain clones from 24-well to be cultured in 6-well in the same Elisa titer high-low and growth state for 5 days, finally selecting less clones from 6-well to be cultured in a shake flask in the same Elisa titer high-low and growth state, freezing a part of the clones safely about 5 days, and remaining for Fed-batch culture.
The control group is Fed-batch culture mother clone sub 1. All subclones used in the assay were screened from the parent sub 1 clone by plating and selecting clones.
Fed-batch culture conditions: inoculating 50ml in a 250ml shake flask, culturing at 36.5 ℃ with the rotation speed of a shaking table of 130rpm, feeding time and times of two groups of experiments are the same, and cooling on day 6.
The Fed-batch culture results are shown in Table 1-Table 2.
TABLE 1 centrifugation Ready-made CHO-S cell semisolid Medium Fed-batch CHO-S cell growth curves
TABLE 2 growth curves for molecular Devices commercial semi-solid medium Clonemedia-CHO-G Fed-batch CHO-S cells
Ten clones were screened by centrifugation from semi-solid culture medium containing CHO-S cells with the highest growth density of 27 × 106cells/ml, the activity rate of most of the clones is more than 50% (14 days), the highest IgG antibody expression titer reaches 3.19G/L, ten clones are screened out by commercial semisolid culture medium CloneMedia-CHO-G, the growth highest density reaches 28 × 106cells/ml, the activity rate is mostly above 50% (14 days), and the highest IgG antibody expression titer reaches 3.25 g/L.
The above test data show that: the application effect of the centrifugal self-prepared CHO-S cell semisolid culture medium provided by the invention basically reaches the application effect of a commercial semisolid culture medium Clonemedia-CHO-G of molecular Devices, and the culture medium can be used for daily production and research and development instead of the culture medium; the preparation method of the semi-solid culture medium provided by the invention is beneficial to improving the application performance of the semi-solid culture medium.
Claims (5)
1. A CHO-S cell semi-solid medium, comprising the following components in percentage by weight: 2.46% of CD FortiCHO AGT Medium Prototype Medium and 2% of methyl cellulose, wherein the viscosity of the methyl cellulose at 20 ℃ is 400cps, and the average molecular weight is 41000;
the CHO-S cell semisolid culture medium is prepared by sufficiently and uniformly mixing a2 XCD FortiCHO AGT mediumPrototype culture medium and 2 Xmethylcellulose according to the volume ratio of 1:1, centrifuging to remove particles invisible to naked eyes, and collecting supernatant centrifugate;
the centrifugation condition is 4000 rpm-10000 rpm, and the centrifugation is carried out for more than 30 min.
2. A method of formulating the CHO-S cell semi-solid medium of claim 1, comprising:
a. fully and uniformly mixing 2 XCD FortiCHO AGT Medium Prototype culture Medium and 2 Xmethylcellulose solution in a volume ratio of 1: 1;
b. centrifuging to remove invisible particulate matters, and collecting supernatant centrifugate to obtain the CHO-S cell semisolid culture medium, wherein the centrifugation condition is 4000 rpm-10000 rpm, and the centrifugation is more than 30 min.
3. The method of claim 2, wherein,
the 2 XCD FortiCHO AGT Medium Prototype culture Medium is prepared according to the following method: weighing CDFortCHO AGT Medium Prototype culture Medium, adding ultrapure water, fully stirring for dissolving, and filtering and sterilizing by a 0.20 mu M filter membrane to obtain 2 multiplied by CD FortCHO AGT Medium Prototype culture Medium;
the 2 x methyl cellulose solution was prepared as follows: weighing methylcellulose, adding ultrapure water, fully stirring and dissolving, and autoclaving to obtain 2 Xmethylcellulose solution.
4. Use of the CHO-S cell semi-solid medium of claim 1 for culturing CHO-S cells.
5. The use according to claim 4, wherein the culture conditions for culturing CHO-S cells are: 5% CO2，36.5℃。
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| CN101663390A (en) * | 2006-09-13 | 2010-03-03 | 艾博特公司 | Cell culture improvements |
| CN102586190A (en) * | 2011-01-11 | 2012-07-18 | 北京华安科创生物技术有限公司 | CHO (Chinese Hamster Ovary) cell strain of efficiently-expressed recombinant human nerve growth factor and construction method thereof |
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